1. Field:
This disclosure is concerned generally with methods of inactivating viruses in solutions of biologically active proteins and, specifically, with the use of azoles to inactivate viruses in an aqueous solution of biologically active therapeutic proteins.
2. Prior Art:
The importance of eliminating viral infectivity in therapeutic products has long been recognized. This is especially true in the case of biologically active products derived from human blood or, more recently, from cell cultures used to make products of biotechnology (e.g., recombinant DNA products and monoclonal antibodies).
In considering virucidal agents for biologically active proteins, the primary goals are to assure complete virucidal action while not adversely affecting the biological activity of the protein. These goals require consideration of such variables as the protein itself, the nature of its activity and/or activity site, the virucidal agent, the importance and/or ease of its removal after use, and variables of the treatment itself, such as time, temperature and concentration.
Heat treatment alone can be used for virucidal treatment of some proteins (e.g., pasteurization at 60.degree. C.). However, it is difficult in many cases to avoid loss of biological activity or utility when heat alone is used.
To avoid some of the disadvantages or activity losses resulting from the use of heat alone, various chemical agents have been used or proposed as virucidal agents for biologically active proteins. See, for example, U.S. Pat. No. 5,071,650, to G. Dove, M. Dobkin and M. Shearer, disclosing the use of alcohols under specific conditions.
The utility of nonexplosive organic solvent/detergent mixtures for the preparation of viral vaccines, and, more recently, for inactivation of endogenous viruses in preparations of biological products derived from human plasma, have been limited by conditions of use (e.g., pH). One virucidal compound used with biologically active proteins at neutral pH is tri-n-butyl phosphate (TNBP). See, for example, U.S. Pat. No. 4,540,573 to A. Neurath and B. Horowitz, disclosing the use of TNBP to inactivate lipid-enveloped viruses. See also U.S. Pat. No. 5,110,910 to G. Tsay disclosing the use of TNBP as a virucide under controlled pH, conductivity and protein concentration. Unfortunately, current virucides typically inactivate mainly lipid-enveloped viruses and have not been shown to be effective against a hardier class of viruses which lack lipid envelopes.
Various azole compounds have been used as fungicides (e.g., see U.S. Pat. No. 5,006,513 to Hector, et al.), but we are unaware of any suggestion to use them as virucides in solutions of biologically active proteins, especially against non-lipid containing viruses. We now have found that azoles provide a unique ability to inactivate both enveloped and non-enveloped viruses in aqueous solutions while preserving the biological activity of proteins such as immunoglobulins. Details of our method are described below.